1. Field of the Invention
The present invention relates to a quantitative analysis film for quantitatively assaying transaminase activity and more particularly to a quantitative analysis film comprising a reagent layer containing a substrate for transaminase, pyruvic acid oxidase and a color indicator composition for detecting hydrogen peroxide, for quantitative assay for transaminase activity.
A method for assaying various transaminase activities which comprise directly producing pyruvic acid by reacting various transaminases with the corresponding substrates, or producing pyruvic acid indirectly through a chemical reaction(s) in which other enzyme(s) participate or through a chemical reaction(s) in which no enzyme participates, reacting the thus produced pyruvic acid with pyruvic acid oxidase and a color indicator composition for detecting hydrogen peroxide which contains peroxidase and then measuring pyruvic acid colorimetrically is known and disclosed in, e.g., Japanese Patent Application (OPI) No. 13068/80. The method as disclosed in this specification involves a series of chemical reactions in an aqueous solution and requires accurate weighing or voluming and complicated handling of an aqueous solution and such accordingly requires long periods of time for analytical operation. Such a method is still insufficient as a method for clinical investigations requiring speed in analysis and accuracy in analytical results.
2. Development of the Invention
Various quantitative analysis films, particularly multi-layered analysis films which permit colorimetric analysis of hydrogen peroxide by dry procedures, have been proposed and some of them have been put in practical use. Among them, there are quantitative analysis films for analysis of glucose, uric acid, cholesterol, choline esterase, creatine, etc., in the living body by a dry procedure which comprises, in sequence, reacting the same with the appropriate oxidizing enzyme or reacting the reaction product formed during an enzyme reaction with the appropriate oxidizing enzyme, reacting the thus released hydrogen peroxide with a color indicator to form color, and then measuring the formed color. In order to enhance the speed of examination, avoid complicated operation and reduce cost, demands for dry type quantitative analysis films have increased, particularly in the field of clinical examination. Thus, quantitative analysis films of the test paper sheet type of the single layer or multi-layer quantitative analysis type of high accuracy in analysis have been developed.
In particular, multi-layer composite type quantitative analysis films are disclosed in Japanese Patent Application (OPI) (the term "OPI" as used herein refers to a "published unexamined Japanese patent application) No. 53888/74 (corresponding to U.S. Pat. No. 3,992,158; hereafter the same have markedly improved accuracy in analysis as compared to conventional quantitative analysis films of the test paper sheet type having a single or dual layers. For purposes of further improving accuracy in analysis and in accordance with the analyte to be assayed, hydrogen peroxide indicators having high detection sensitivity have also been provided in such multi-layer composite type quantitative analysis forms; examples of indicators for detecting hydrogen peroxide and layer structures for such multi-layer analysis films are disclosed in Japanese Patent Applications (OPI) Nos. 40191/76 (corresponding to U.S. Pat. No. 4,042,335), 131089/78, 29700/79 (corresponding to U.S. Pat. No. 4,166,093), 124499/80, etc.
Indicators for detecting hydrogen peroxide used for these quantitative analysis films by a dry procedure are based on those for detecting hydrogen peroxide employed in conventional quantitative analysis by a dry procedure and the principles are the same.
The normal range for transaminase values in a normal human is approximately 20 IU/l at maximum in whole blood and an amount of a substrate which undergoes a catalytic action of transaminase during a time period for several ten minutes required for measurement methods conventionally employed is a concentration of 10.sup.-4 mole/l even under optimal conditions. For effecting a colorimetric measurement of such a trace amount of a substrate through coupled enzyme reactions, it is required that: (1) respective reactions sufficiently proceed, (2) light extinction of a dye formed is large, (3) a dye is stable until it is measured and (4) in the case where a colorimetric measurement is performed using a quantitative analysis film, a produced dye is not lost by the dye being mobilized or diffused until it is measured. As a result of extensive investigations, it has been found that by selecting such a system that the reaction product is converted into hydrogen peroxide via pyruvic acid by transaminase enzyme reaction or a combination of transaminase enzyme reaction with other enzyme reaction(s) coupled with transaminase enzyme reaction, by selecting as a color indicator system for detecting hydrogen peroxide such a system that a cationic dye is produced and by the use of 4-aminoantipyrine or an N,N-disubstituted-p-phenylenediamine as a hydrogen donor and an N,N-disubstituted aniline derivative as a coupler in combination, high reaction efficiency, large light extinction of a produced cationic dye and markedly high sensitivity in transaminase activity measurement are obtained.